Summary Tuberculosis (TB) is a major global health problem. Bacille de Calmette et Gurin (BCG), the only TB prophylactic vaccine licensed, is 80% effective against extra-pulmonary TB (extra-PTB), but has variable efficacy against PTB, the site of primary TB infection. A big handicap in the development of TB vaccines more effective than BCG is that immune protection against TB is incompletely understood. BCG generates TB- specific adaptive cell-mediated immunity (aCMI), which is thought to contribute especially to its strong protective effect against extra-PTB, but does not confer sterile immunity against M. tuberculosis (Mtb) and does not protect against latent TB infection (LTBI). We hypothesized that Mtb memory-like innate CMI (iCMI) is a mechanism of protection against primary Mtb infection that prevents LTBI. An important corollary is that TB- specific iCMI will provide a robust measure for vaccine-induced sterile protection against TB, a much needed tool for the development of highly efficacious TB vaccines. Using samples collected in the Cohort For TB Research By The Indo-US Medical Partnership Multicentric Prospective Observational Study (C-TRIUMPH) at the Byramjee Jeejeebhoy Govt. Medical College (BJMC), Pune, India, together with a group of BCG recipients with negligible exposure to TB to be enrolled in the US, we will investigate TB-specific iCMI as a mechanism of sterile protection against Mtb, and the extent to which Mtb memory iCMI can be elicited by BCG vaccination. AIM 1. To identify the Mtb iCMI characteristics that differentiate LTBI- from LTBI+ adults with high exposure to smear-positive PTBI. Hypothesis: LTBI- individuals highly exposed to TB have more robust Mtb memory-like iCMI than LTBI+ individuals. Using CyTOF technology and systems biology analytical tools designed for large datasets, we will measure a large array of NK, NKT, ?? T and mucosal-associated invariant T (MAIT) cell responses to ex-vivo Mtb antigenic stimulation and analyze the differences between a subset of LTBI- and LTBI+ adults highly exposed to smear-positive PTBI. The most significant independent differences will be used to build a smaller flow cytometry panel that will be used to test the remaining LTBI- and LTBI+ highly TB-exposed adults. AIM 2. To characterize the Mtb-specific iCMI generated by BCG. Hypothesis: Compared with LTBI- individuals highly exposed to TB, BCG administration generates memory-like iCMI to Mtb of lower magnitude and/or restricted to a fraction of the vaccine recipients. We will recruit adults who received BCG and had negligible exposure to TB and compare their Mtb iCMI with that of highly TB-exposed LTBI- adults using the tools described in AIM 1. The results of this study have the potential to shift the paradigm for immune protection against Mtb infection by substituting and/or adding iCMI to aCMI as long term protective responses and, thereby, to revolutionize the field of TB vaccine development. The study will provide added value to C-TRIUMPh by using already collected peripheral blood mononuclear cells (PBMC) to accomplish its immunologic goals. We will also leverage the NIH efforts to develop advanced immunologic capacity at BJMC.